The field of the present invention relates to the detection of controlled substances. In particular, it relates to labeled tracers for use in detecting cannabinoids and tetrahydrocannabinoids (THC) in biological samples.
Marijuana, a known psychoactive drug, is derived from plants of the hemp family that produce significant amounts of cannabinoids. In particular, the most important cannabinoid is xcex949-tetrahydrocannabinol (xcex949-THC), the major physiologically active constituent of marijuana. xcex949-THC is a controlled substance because it has both sedative and depressant-like effects on the cardiovasular and central nervous systems, as opposed to cannabidiol, a non-psychoactive constituent of marijuana. Through smoking marijuana, xcex949-THC is rapidly absorbed from the lungs into the blood stream and metabolized through 11-nor-xcex949-THC to a series of polar metabolites with 11-nor-xcex949-THC-carboxylic acid as the primary metabolite.
Due to the common abuse of cannabinoids, there is a growing need for non-invasive and rapid tests to detect the presence of these controlled drugs in biological specimens. Currently, cannabinoids in biological samples can be detected by a number of techniques such as thin layer chromatography (TLC), gas chromatography/mass spectrometry (GC/MS), radioimmunoassay or enzyme immunoassay. Depending upon assay sensitivity, cannabinoid metabolites may be detected in the urine for up to 10 days in occasional smokers and 36 days in chronic smokers. See Wang et al, U.S. Pat. No. 5,264,373, col 1, lines 36-37.
In recent years, there have been many reports concerning the use of saliva for drug monitoring. Saliva testing for the presence of xcex949-THC has been applied to pharmacokinetic studies or to the management of patients in chronic drug therapy. See Samyn, et al, Forensic Science Review, vol. 11, p. 1, (1999). Although saliva testing does not quantify the blood concentration of the drug, (See Gross et al, xe2x80x9cValidated Direct Blood 9-THC Radioimmunoassay Quantitationxe2x80x9d, J. Anal. Toxi. Vol.2, p.98 (1978)), saliva testing is of particular interest since the presence of xcex949-THC in the saliva may indicate recent smoking of cannabinoids. The concentration of xcex949-THC in the blood may also be estimated from the concentration of xcex949-THC; the blood concentration is usually much higher than the drug concentration in saliva. Idowu, et al, xe2x80x9cA Review of the Use of Saliva in the Forensic Detection of Drugs and Other Chemicalsxe2x80x9d, J. Forensic Science Society, v22, 1982, p123.
Furthermore, the use of a continuous flow displacement immunoassay technology (F. S. Ligler, et al, Flow Immunosensor Method and Apparatus, U.S. Pat. No. 5,183,740) has been demonstrated for rapid detection of controlled drugs in saliva and urine. See Hao Yu et al, Use of the USDT Flow Immunosensor for Quantitation of Benzolecgonie in Urine, Biosensors and Bioelectronics, 732-734(1996); Nam, D. et al. Programme and Abstracts of TIAFT 2000 at Helsinki, 2000; Liang, G. et al., Proc. of ICADTS 2000, Jun. 22-26, 2000. U.S. Pat. No. 5,183,740 and the above cited references, including any figures contained therein, are hereby incorporated by reference as if fully set forth herein.
To detect xcex949-THC using an immunoassay or immunosensor, a tracer molecule is usually used to compete with xcex949-THC or its metabolites. The tracer molecule is usually a labeled antigen or ligand, capable of binding to the same antigen or ligand binding site(s) of an antibody or receptor to xcex949-THC or its metabolites. In detecting controlled substances, most immunoassays have generally used the labeled illicit drugs themselves, (e.g., labeled xcex949-THC) as tracers to detect the presence and/or to quantify the analytes in the sample.
Recent use of non-controlled substances as starting materials in xcex949-THC tracers synthesis has also been reported by Wang, et al, in U.S. Pat. No. 5,264,373 entitled Fluorescence polarization Immunoassay for tetrahydrocannabinoids. In this patent, Wang discloses the use of fluorescein to label THC-analog based derivatives for use in a fluorescence polarization immunoassay.
FIG. 1 generally depicts the various methods for synthesizing tracers used in the detection of xcex949-THC or it metabolites. Panel A, for example, depicts one of the common methods that use controlled substances, such as the illicit drug, 9-carboxy (or aldehyde)-xcex949-THC, as starting materials. These starting materials are coupled with labels at the carboxyl group attached to the carbon at position 9 on xcex949-THC to yield a drug-based tracers. Panel B depicts an alternative method of synthesizing a tracer, which uses xcex949-THC-analogs.
In certain immunoassay systems such as a continuous flow displacement assay, it is desirable that the binding affinity of the antibody to the tracer molecules be lower than the binding affinity of the antibody to the analyte to facilitate effective displacement. Using a xcex949-THC-based or xcex949-THC-analog based tracers is problematic because the xcex949-THC analyte does not effectively displace the xcex949-THC -based or xcex949-THC -analog based tracer. This results in a lower sensitivity of the assay.
Hence, there exists a need for novel reagents, in particular, novel tracers for use in detecting xcex949-THC.
The present invention provides a novel set of tracers produced from coupling cannabinol (6,6,9-trimethyl-3-pentyl-6H-dibenzo[b,d]pyran-1-ol) with a label to yield cannabinol-based tracers. In one aspect of the invention, the label can be attached at positions 1, 2, 3, 4, 8, 9, and 10 of the cannabinol molecule.
In another aspect of the invention, the cannabinol-based tracer can be used in conjunction with recognition molecules that are capable of binding to cannabinoids such as xcex949-THC or its metabolites. Examples of these recognition molecules include antibody or receptor molecules that are capable of binding cannabinoids.
In a preferred embodiment of the invention, the cannabinol-based tracers are used in conjunction with a continuous flow displacement assay system. In this embodiment, it is preferred that the antibody or receptor molecule binds to the cannabinol-based tracer at a lower binding affinity than the affinity to the cannabinoids (e.g., xcex949-THC or its metabolites) present in the sample.